Methods and compositions for producing novel conjugates of thrombin inhibitors and endogenous carriers resulting in anti-thrombins with extended lifetimes

ABSTRACT

Novel compounds comprising chemically reactive intermediates which can react with available reactive functionalities on blood components to form covalent linkages, where the resulting covalently-bound conjugates are found to have thrombin inhibition activity are provided. Specifically, the thrombin inhibitor compounds of the present invention are derivatives of the known thrombin inhibitor argatroban, which can be covalently linked to chemically reactive functionalities on various blood components. The conjugated thrombin inhibitors thereby have extended lifetimes in the bloodstream, as compared to the unconjugated parent drug, and are, therefore, capable of maintaining thrombin inhibitory activity for extended periods of time as compared to the unconjugated parent drug. Also provided herein are methods for inhibiting thrombin activity in vivo comprising administering to the bloodstream of a mammalian host the novel compounds of the present invention.

INTRODUCTION

1. Technical Field

The field of this invention is the extended lifetime of physiologicallyactive agents in a mammalian host, more specifically, the extendedlifetime of inhibitors of thrombin activity in a mammalian host.

2. Background

In the past, there have been many attempts to obtain new and improvedagents for the treatment of thrombosis. The N²-(p-tolysulfonyl)-L-arginine esters are one type of agent which can beused and these have been found to be effective in dissolving blood clots(see U.S. Pat. No. 3,622,615, issued Nov. 23, 1971). Another family ofcompounds which have been found to be particularly useful as highlyspecific inhibitors of thrombin for the control of thrombosis is the N²-dansyl-L-arginine esters or amides (U.S. Pat. No. 3,978,045) and manyN² -arylsulfonyl-L-argininamides. Moreover, another compound which hasproven to be particularly useful for the treatment of thrombosis inmammals is 1- 5- Aminoiminomethyl)amino!-1-oxo-2-(1,2,3,4-tetrahydro-3-methyl-8-quinolinyl)sulfonyl!-amino!pentyl!-4-methyl-2-piperidinecarboxylicacid, a compound which is commonly known as argatroban.

Although the above described compounds have proven useful for thetreatment of disorders associated with abnormal thrombosis viainhibition of thrombin activity in vivo, their therapeutic utility issomewhat limited due to the fact that many of these compounds arequickly degraded and/or removed from the host's vascular system afteradministration. As such, for continuous thrombin inhibitory activityover extended periods of time, the above described thrombin inhibitorsmust be administered as a large bolus given at periodic time intervalsor by providing depots comprising the drug. Unfortunately, however,administration of large boluses at periodic time intervals often resultsin subtherapeutic doses of the drug for extended periods of timefollowed by doses which may greatly exceed the desired therapeuticlevel. The latter may often involve serious adverse side effects.Moreover, although various pumps and biodegradable and non-biodegradablecapsules have been devised for the delivery of drug over an extendedperiod of time, these devices may have a variety of shortcomings intheir profile of drug delivery, for example, often resulting in aninflammatory response and/or being subject to interference in theirrelease of active drug.

There is, therefore, an interest in providing improved therapiesassociated with thrombin inhibition.

SUMMARY OF THE INVENTION

Novel compounds are provided comprising chemically reactiveintermediates which can react with available reactive functionalities onblood components to form covalent linkages, where the resultingcovalently-bound conjugates are found to have thrombin inhibitionactivity. Specifically, the compounds of the present invention arechemically reactive derivatives of the known thrombin inhibitor 1- 5-Aminoiminomethyl)amino!-1-oxo-2-(1,2,3,4-tetrahydro-3-methyl-8-quinolinyl)sulfonyl!-amino!pentyl!-4-methyl-2-piperidinecarboxylicacid, a compound also known as argatroban. The derivatized argatrobanmolecules of the present invention comprise an active thrombin inhibitorportion and become covalently linked through a chemically reactive groupto reactive functionalities on various blood components. The conjugatedthrombin inhibitor molecules thereby have extended lifetimes in thebloodstream, as compared to the unconjugated parent drug, and are,therefore, capable of maintaining thrombin inhibitory activity forextended periods of time as compared to the unconjugated parent drug.

Also provided herein are compositions comprising the derivatizedthrombin inhibitor molecules described above combined withpharmaceutically acceptable carriers and methods for inhibiting thrombinactivity in vivo comprising administering to the bloodstream of amammalian host the novel compounds of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of the steps involved in the synthesisof various derivatized thrombin inhibitor molecules of the presentinvention.

FIG. 2 is a graph showing the results obtained in sandwich ELISA assayswith human serum albumin (HSA) alone (""), HSA treated with prequenchedhydroxylamine treated argatroban-C6 NHS ester ("Δ"), HSA treated withprequenched argatroban-C12 NHS ester ("*"), HSA reacted withargatroban-C6 NHS ester ("♦") or HSA reacted with argatroban-C12 NHSester ("X"). Results are presented as the absorbance at 490 nm ("OD490") versus HSA concentration in nanograms per milliliter ("ng/ml").

FIG. 3 is a graph showing the results obtained in sandwich ELISA assayswith rabbit serum albumin (RSA) alone ("Δ"), RSA reacted withprequenched argatroban-C6 NHS ester ("▪") or RSA reacted withargatroban-C6 NHS ester ("♦"). Results are presented as the absorbanceat 490 nm ("OD 490") versus RSA concentration in nanograms permilliliter ("ng/ml").

FIG. 4 is a graph showing the results obtained in sandwich ELISA assayswith human glycophorin A (hGPA) alone ("Δ"), hGPA treated withprequenched argatroban-C6 NHS ester ("▪") or hGPA treated withargatroban-C6 NHS ester ("♦"). Results are presented as the absorbanceat 490 nm ("OD 490") versus hGPA concentration in nanograms permilliliter ("ng/ml").

FIG. 5 is a graph showing the results obtained in sandwich ELISA assayswith HSA alone ("▪"), HSA treated with prequenched argatroban-C6 NHSester ("*"), HSA treated with argatroban-C6 NHS ester (1:500 dilution)("♦") or HSA reacted with argatroban-C6 NHS ester (1:1000 dilution)("X"). Results are presented as the absorbance at 490 nm ("OD 490")versus HSA concentration in nanograms per milliliter ("ng/ml").

FIG. 6 is a graph showing the results obtained in sandwich ELISA assayswith hGPA alone ("Δ"), hGPA treated with prequenched argatroban-C6 NHSester ("▪") or hGPA reacted with argatroban-C6 NHS ester ("♦") employingthe 5F4 monoclonal antibody. Results are presented as the absorbance at490 nm ("OD 490") versus hGPA concentration in nanograms per milliliter("ng/ml").

FIG. 7 is a graph showing the results obtained in sandwich ELISA assayswith hGPA alone ("Δ"), hGPA treated with prequenched argatroban-C6 NHSester ("▪") or hGPA reacted with argatroban-C6 NHS ester ("♦") employingthe 3C10 monoclonal antibody. Results are presented as the absorbance at490 nm ("OD 490") versus hGPA concentration in nanograms per milliliter("ng/ml").

FIG. 8 is a graph showing the results obtained in sandwich ELISA assayswith hGPA alone ("Δ"), hGPA treated with prequenched argatroban-C6 NHSester ("▪") or hGPA reacted with argatroban-C6 NHS ester ("♦") employingthe 3E4 monoclonal antibody. Results are presented as the absorbance at490 nm ("OD 490") versus hGPA concentration in nanograms per milliliter("ng/ml").

FIG. 9 is a graph showing the results obtained in thrombin inhibitorassays with RSA alone ("α") or RSA reacted with argatroban-C6 NHS ester(""). Results are presented as the % inhibition of thrombin versus theamount of RSA employed in nM.

FIG. 10 is a graph showing the results obtained in thrombin inhibitorassays with thrombin alone ("∘") or erythrocyte ghosts reacted withargatroban-C6 NHS ester (""). The points on the graph wherein conjugatederythrocyte ghosts were employed are each shown by the symbol (""),however, those points define 3 parallel lines representing (from the topof the graph to the bottom) the addition of 200 μg, 450 μg and 600 μg ofghosts/ml. Results are presented as the thrombin activity versus thenumber of minutes at 21° C.

FIG. 11 is a graph showing the results obtained demonstrating theability of the novel thrombin inhibitors of the present invention, whencovalently bound to RSA, to delay thrombin-induced platelet aggregation.Data is presented as the concentration in μg/ml of RSA alone ("RSA") orRSA covalently bound by argatroban-C6 NHS-ester molecules ("RSA--Ar")versus the time in minutes to achieve 50% platelet aggregation. Assayswere run in duplicate and each assay is shown as a separate column ateach concentration tested.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions are provided for the treatment of patientshaving disorders associated with abnormal thrombosis through inhibitionof the mammalian protein, thrombin. The method employs a novel thrombininhibitor compound comprising a derivative of the known thrombininhibitor argatroban, wherein the novel thrombin inhibitor compound hasa chemically reactive group which reacts with available reactivefunctionalities on various blood components, thereby covalently bondingthe derivatized thrombin inhibitor molecule to those blood components.The chemically reactive group is at a site, so that when the thrombininhibitor portion is bonded to the blood component, the thrombininhibitor portion retains a substantial proportion of the parentcompound's inhibitory activity. Therefore, through the covalent bondingof the subject thrombin inhibitor molecules to long-lived bloodcomponents, the effective lifetime of the thrombin inhibitor in thehost's vascular system is greatly increased.

The various sites with which the chemically reactive group of thesubject thrombin inhibitor molecules may react include cells,particularly red blood cells (erythrocytes) and platelets, and proteins,such as immunoglobulins, including IgG and IgM, serum albumin, ferritin,steroid binding proteins, transferrin, thyroxin binding protein,α-2-macroglobulin, and the like. Those proteins with which thederivatized thrombin inhibitor compounds react, which are notlong-lived, will generally be eliminated from the host within aboutthree days, so that the proteins indicated above (including the proteinsof the cells) will remain at least three days, usually at least fourdays, and may remain five days or more, usually not exceeding 60 days,more usually not exceeding 30 days, particularly as to the half life,based on the concentration in the blood, as measured in from about 1-3hours after administration.

For the most part, reaction will be with mobile components in the blood,particularly blood proteins and cells, more particularly blood proteinsand erythrocytes. By "mobile" is intended that the component does nothave a fixed situs for any extended period of time, generally notexceeding 5, more usually one minute, although some of the bloodcomponent may be relatively stationary for extended periods of time.Initially, there will be a relatively heterogeneous population offunctionalized proteins and cells. However, for the most part, thepopulation within a few days will vary substantially from the initialpopulation, depending upon the half-life of the functionalized proteinsin the blood stream. Therefore, usually within about three days or more,IgG will become the predominant functionalized protein in the bloodstream.

Usually, by day 5 post-administration, IgG, serum albumin anderythrocytes will be at least about 60 mole %, usually at least about 75mole %, of the conjugated components in blood, with IgG, IgM (to asubstantially lesser extent) and serum albumin being at least about 50mole %, usually at least about 75 mole %, more usually at least about 80mole %, of the non-cellular conjugated components.

The derivatized thrombin inhibitor molecules of the present inventionwill, for the most part, have the following formula: ##STR1##

wherein:

Y is a linking group having from 2-30, more usually from 2-18,preferably from 6-12 atoms in the chain, particularly carbon, oxygen,phosphorous and nitrogen, more particularly carbon and oxygen, where Ymay be alkylene, oxyalkylene, or polyoxyalkylene, preferably an alkylchain having from 2-15, more preferably from 6-12 carbon atoms in thealkyl chain, and the like; and

Z is a chemically reactive group or precursor to a chemically reactivegroup, such as carboxy, carboxy ester, where the ester group is of 1-8,more usually 1-6 carbon atoms, particularly a physiologically acceptableleaving group which activates the carboxy carbonyl for reaction withamino groups in an aqueous system, e.g. N-hydroxysuccinimide,isocyanate, thiolester, thionocarboxylic acid ester, imino ester, mixedanhydride, e.g. carbodiimide anhydride, carbonate ester, phosphorylester, etc. and the like.

The activatable precursor will usually be a non-oxo-carbonyl group,including the sulfur and nitrogen analogs thereof, such as thiono andthiol acids and esters and imino esters or functionalities which can bedirectly modified to provide an active functionality, e.g. cyano.

The reactive functionalities which are available on proteins forcovalently bonding to the chemically reactive group of the derivatizedthrombin inhibitors of the present invention are primarily amino groups,carboxyl groups and thiol groups. While any of these may be used as thetarget of the chemically reactive group on the thrombin inhibitor, forthe most part, bonds to amino groups will be employed, particularly withthe formation of amide bonds. To form amide bonds, one may use as achemically reactive group a wide-variety of active carboxyl groups,particularly esters, where the hydroxyl moiety is physiologicallyacceptable at the levels required. While a number of different hydroxylgroups may be employed, the most convenient will be N-hydroxysuccinimide(NHS), and N-hydroxy sulfosuccinimide (sulfo-NHS), although otheralcohols, which are functional in an aqueous medium such as blood, mayalso be employed. In some cases, special reagents find use, such asazido, diazo, carbodiimide anhydride, hydrazine, dialdehydes, thiolgroups, or amines to form amides, esters, imines, thioethers,disulfides, substituted amines, or the like. Usually, the covalent bondwhich is formed should be able to be maintained during the lifetime ofthe blood component, unless it is intended to be a release site.

If desired, the subject conjugates may also be prepared in vitro bycombining blood with derivatized thrombin inhibitors of the presentinvention, allowing covalent bonding of the derivatized thrombininhibitors to reactive functionalities on blood components and thenreturning the conjugated blood to the host. Moreover, the above may alsobe accomplished by first purifying an individual blood component orlimited number of components, such as red blood cells, immunoglobulins,serum albumin, or the like, and combining the component or components invitro with the chemically reactive thrombin inhibitors. Thefunctionalized blood or blood component may then be returned to the hostto provide in vivo the subject therapeutically effective conjugates. Theblood also may be treated to prevent coagulation during handling invitro.

When conjugates are prepared in vitro, the ratio of derivatized thrombininhibitor to blood components will vary widely, depending upon whetherwhole blood or just a purified component is used as a bonding site forthe derivatized thrombin inhibitor. For reacting with whole blood, onewill normally have a ratio of derivatized thrombin inhibitor to blood ofabout 38 micrograms per ml to about 300 micrograms per ml, respectively,while the ratio of derivatized thrombin inhibitor to 10⁹ cells will bein the range of about 7.6 micrograms to about 300 micrograms, while theratio of derivatized thrombin inhibitor to 1 mg of protein will be inthe range of about 38 micrograms to about 300 micrograms.

The nature of the thrombin inhibitor compound may provide for randombonding to the long lived blood components or, to varying degrees,targeted bonding to a restricted class of blood components. For randombonding, the distribution of the thrombin inhibitor compound will bebased on the relative proportion of active sites for bonding of theblood components, the mode of administration and whatever preferentialassociation the thrombin inhibitor may have. For targeted bonding, apart of the thrombin inhibitor compound will be a group whichpreferentially non-covalently binds to one or more sites present on oneor more of the blood components. For this purpose, entities which areknown to complex with particular blood, components may be used.Alternatively, one may prepare a combinatorial library and screen formembers of the library which provide the desired blood componentassociation spectrum. For the most part, random bonding will beemployed.

To the extent that targeted bonding is employed, the choice of the longlived blood component will be affected, at least in part, by the desiredlifetime for the drug and the availability of the blood component forbonding to the derivatized thrombin inhibitor.

A long lived blood component has a half life of at least about 12 hours,usually at least about 48 hours, preferably at least about 5 days,desirably at least about 10 days and more desirably at least about 20days or more. Generally, half lives are determined by serialmeasurements of whole blood, plasma or serum levels of the compoundfollowing labeling of the compound with an isotope (e.g. ¹³¹ I, ¹²⁵ I,Tc, 5¹ Cr ³ H, etc.) or fluorochrome and injection of a known quantityof labeled compound intravascularly. Included are red blood cells (halflife ca. 60 days), platelets (half life ca. 4-7 days), endothelial cellslining the blood vasculature, and long lived blood serum proteins, suchas albumin, steroid binding proteins, ferritin, α-2-macroglobulin,transferrin, thyroxin binding protein, immunoglobulins, especially IgG,etc. In addition to preferred half lives, the subject components arepreferably in cell count or concentration sufficient to allow binding oftherapeutically useful amounts of the compound of the present invention.For cellular long lived blood components, cell counts of at least2,000/μl and serum protein concentrations of at least 1 μg/ml, usuallyat least about 0.01 mg/ml, more usually at least about 1 mg/ml, arepreferred.

The cellular long lived blood components to which the subjectderivatized thrombin inhibitors bond are present in high number in thevascular system. Platelets are present in from about 1-4×10⁵ /μl, whilered blood cells are present in about 4-6×10⁶ /μl. The cells have a longhalf life and the binding to a surface membrane protein of the cellsappears not to result in endocytosis. Preferred cells have a widedistribution in capillaries and tissue and express specific bindingsites on the cell surface associated with their specificdifferentiation. In addition to in vivo administration of the subjectderivatized thrombin inhibitors, in the case of red blood cells andplatelets, these cells may be readily collected, combined with theconjugate in vitro, and then administered to the host. The cells willnormally be autologous or allogeneic, but in some instances may even bexenogeneic.

Suitable erythrocyte binding site containing molecules includeglycophorin A, B and C, Band 3 and Rhesus blood group antigens.Preferred erythrocyte binding sites are abundantly expressed on theerythrocyte with copy numbers of at least 1,000, preferably at least10,000, more preferably at least 100,000 per cell, desirably aretethered at least about 0.5, preferably at least about 1 nm above thebilayer surface and do not facilitate per se cell deformation when thederivatized thrombin inhibitor molecule is bound to the cell (e.g. thebinding will be selected so as not to be a key component of thecytoskeleton). Binding sites of the erythrocyte surface glycoproteinglycophorin A and erythrocyte binding sites comprising sialic acid areexamples of preferred binding sites. Preferred platelet binding sitesinclude GPIIa, GPIIb, GPIIIa and GPIV. Desirably, upon bonding to thetarget, deformation of the long-lived blood component, e.g. erythrocyteor platelet, does not occur.

The derivatized thrombin inhibitors of the present invention willusually be administered as a bolus, but may be introduced slowly overtime by infusion using metered flow, or the like. Alternatively,although less preferable, blood may be removed from the host, treated exvivo, and returned to the host. The derivatized thrombin inhibitors willbe administered in a physiologically acceptable medium, e.g. deionizedwater, phosphate buffered saline (PBS), saline, aqueous ethanol or otheralcohol, plasma, proteinaceous solutions, mannitol, aqueous glucose,alcohol, vegetable oil, or the like. Other additives which may beincluded include buffers, where the media are generally buffered at a pHin the range of about 5 to 10, where the buffer will generally range inconcentration from about 50 to 250 mM, salt, where the concentration ofsalt will generally range from about 5 to 500 mM, physiologicallyacceptable stabilizers, and the like. The compositions may belyophilized for convenient storage and transport.

The subject derivatized thrombin inhibitors will for the most part beadministered parenterally, such as intravascularly (IV), intraarterially(IA), intramuscularly (IM), subcutaneously (SC), or the like.Administration may in appropriate situations be by transfusion. In someinstances, where reaction of the active functional group is relativelyslow, administration may be oral, nasal, rectal, transdermal or aerosol,where the nature of the conjugate allows for transfer to the vascularsystem. Usually a single injection will be employed although more thanone injection may be used, if desired. The derivatized thrombininhibitors may be administered by any convenient means, includingsyringe, trocar, catheter, or the like. The particular manner ofadministration will vary depending upon the amount to be administered,whether a single bolus or continuous administration, or the like.Preferably, the administration will be intravascularly, where the siteof introduction is not critical to this invention, preferably at a sitewhere there is rapid blood flow, e.g. intravenously, peripheral orcentral vein. Other routes may find use where the administration iscoupled with slow release techniques or a protective matrix. The intentis that the subject compound be effectively distributed in the blood, soas to be able to react with the blood components. The concentration ofthe conjugate will vary widely, generally ranging from about 1 pg/ml to50 mg/ml. The total administered intravascularly will generally be inthe range of about 0.1 mg/ml to about 10 mg/ml, more usually about 1mg/ml to about 5 mg/ml.

The manner of producing the derivatized thrombin inhibitors of thepresent invention will vary widely, depending upon the nature of thevarious elements comprising the molecule. The synthetic procedures willbe selected so as to be simple, provide for high yields, and allow for ahighly purified product. Normally, the chemically reactive group will becreated as the last stage, for example, with a carboxyl group,esterification to form an active ester will be the last step of thesynthesis. An illustrative method for the production of the derivatizedthrombin inhibitors of the present invention is shown in FIG. 1.

By bonding to long-lived components of the blood, such asimmunoglobulin, serum albumin, red blood cells and platelets, a numberof advantages ensue. The inhibition of the thrombin protein is extendedfor days to weeks. Only one administration need be given during thisperiod of time. Greater specificity can be achieved, since the activecompound will be primarily bound to large molecules, where it is lesslikely to be taken up intracellularly to interfere with otherphysiological processes.

The blood of the mammalian host may be monitored for the presence of thethrombin inhibitor one or more times. By taking a portion or sample ofthe blood of the host, one may determine whether the thrombin inhibitorhas become bound to the long lived blood components in sufficient amountto be therapeutically active and, thereafter, the level of thrombininhibition activity in the blood. If desired, one may also determine towhich of the blood components the thrombin inhibitor molecule is bound.

The derivatized thrombin inhibitors of the present invention will finduse in a variety of different applications. For example, the novelthrombin inhibitors of the present invention are useful for thetreatment and/or prevention of disorders associated with abnormalthrombosis. The subject thrombin inhibitor molecules are useful forinhibiting the production of thromboses, accelerating the dissolution ofexisting thromboses and for maintaining and/or improving bloodcirculation. The subject derivative molecules will also find use, forexample, for treatment after surgery, after myocardial infarction,arteritis of the legs, deep venous thrombosis, pulmonary embolism, andthe like.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Synthesis of Extended Lifetime DerivatizedThrombin Inhibitor Molecules

Materials and Methods: The synthesis of various derivatized extendedlifetime thrombin inhibitor (argatroban-derived) molecules isschematically diagramed in FIG. 1 and is described below.

(1) Synthesis of Methyl 12-Aminododecanoate (compound YS-004-17).

To a suspension of 12-aminododecanoic acid (5.00 g, 23.3 mmol) inanhydrous MeOH (90 mL) was introduced hydrogen chloride gas for 25minutes, during which time the suspension became clear. The solution wasthen stirred at room temperature for 3 hours and the solvent was removedin vacuo. The white solid residue was dissolved in H₂ O. Solid NaHCO₃was added to neutralize the solution to pH 8-9. The white precipitatesthus formed were collected by vacuum filtration (5.20 g) (Yield: 98%).

¹ HNMR (MeOH-d4, 300 MHz) δ 3.67 (s, 3H, OCH₃), 2.68 (t, 2H, J=7.1 Hz,NCH₂), 2.30 (t, 2H, J=7.5 Hz, CH₂ CO₂), 1.68-1.56 (m, 2H, CH₂),1.50-1.38 (m, 2H, CH₂), 1.27 (br. s, 12 H, (CH₂)₆), 1.20-0.90 (m, 2H,CH₂).

(2) Synthesis of Methyl 6-Aminohexanoate (compound YS-004-58).

To a suspension of 6-aminocaproic acid (3.00 g, 22.9 mmol) in anhydrousMeOH (60 mL) was introduced hydrogen chloride gas for 25 minutes, duringwhich time the suspension became clear. The solution was then stirred atroom temperature for 5 hours and MeOH was removed in vacuo. The residuewas recrystallized from THF to give a white solid (3.10 g). (Yield:75%).

¹ HNMR (MeOH-d4, 300 MHz) δ 3.66 (s, 3H, OCH₃), 2.92 (t, 2H, J=7.7 Hz,NCH₂), 2.37 (t, 2H, J=7.2 Hz, CH₂ CO₂), 1.74-1.60 (m, 4H, CH₂ CH₂),1.48-1.36 (m, 2H, CH₂)

3. Synthesis of Argatroban-C12 Methyl Ester (compound YS-004-67).

To a solution of argatroban monohydrate (300 mg, 0.570 mmol) and methyl12-aminododecanoate (compound YS-004-17) (132 mg, 0.576 mmol) inanhydrous DMF (15 mTL) was added HBTU (258 mg, 0.680 mmol). Stirring wascontinued at room temperature for 16 hours. The precipitates formedduring the reaction were filtered off and DMF was removed by vacuumdistillation. The residue was purified by preparative TLC using solventsCH₂ Cl₂ /MeOH/NH₃ (90/10/1, v/v) to give the titled compound (140 mg)(Yield: 28%). ¹ HNMR (MeOH-d4, 300 MHz).

4. Synthesis of Argatroban-C6 Methyl Ester (compound YL-015-13).

To a solution of argatroban monohydrate (100 mg, 0.190 mmol) and methyl6-aminohexanoate hydrochloride (compound YS-004-58) (35 mg, 0.193 mmol)in anhydrous DMF (5 ml) was added triethylamine (23 mg, 0.23 mmol),followed by addition of HBTU (86 mg, 0.23 mmol). Stirring was continuedat room temperature for 20 hours. The precipitates formed during thereaction were filtered off and DMF was removed by vacuum distillation.The residue was purified by preparative TLC using solvents CH₂ Cl₂/MeOH/NH₃ (90/10/1, v/v) to give the titled compound (78 mg) (Yield:53%). ¹ HNMR (MeOH-d4, 300 MHz).

5. Synthesis of Argatroban-C12 Free Acid (compound YL-015-10). A

A solution of compound YS-004-67 (225 mg, 0.260 mmol) in MeOH (4.2 mL)and 1M aqueous NaOH (0.87 mL) was stirred at room temperature for 24hours. MeOH was removed in vacuo to give a cloudy solution, to whichmore H₂ O was added until the solution became clear. The solution wasacidified with 1N HCl to pH 3.0 while being cooled at 0° C. to give awhite precipitate, which was collected by vacuum filtration to affordthe titled compound (105 mg). The filtrate was extracted with n-butanol(4×4 mL). The combined butanol solution was washed with H₂ O (to pH 5.0)and concentrated in vacuo azeotropically with H₂ O to give anotherportion of the compound (58 mg) (Yield: 85%). ¹ HNMR (MeOH-d4, 300 MHz).

6. Synthesis of Argatroban-C6 Free Acid (compound YL-015-15).

A solution of compound YL-015-13 (124 mg, 0.159 mmol) in MeOH (3mL) and1M aqueous NaOH (0.55 mL) was stirred at room temperature for 33 hours.The reaction was worked up by the same procedure as described for thepreparation of compound YL-015-10, to give the titled compound (76 mg)(Yield: 73%). ¹ HNMR (MeOH-d4, 300 MHz).

7. Synthesis of Argatroban-C12 NHS Ester (compound YL-015-11).

To a solution of compound YL-015-10 (40 mg, 0.054 mmol) andN-hydroxysuccinimide (13 mg, 0.11 mmol) was added diisopropylethylamine(7.4 mg, 0.057 mmol), followed by addition of HBTU (43 mg, 0.11 mmol).The reaction mixture was stirred at room temperature for 36 hours. DMFwas removed by vacuum distillation and the residue was dissolved in MeOH(4 mL). The MeOH solution was filtered to remove the insolubles, thefiltrate was concentrated in vacuo, and the residue was dissolved in aminimum amount of MeOH. H₂ O was then added to induce precipitation andthe precipitate was dried on vacuum to give the titled compound (19 mg)(Yield: 42%).

The yield of the reaction was later improved by using EDC as thecoupling reagent, as exemplified below. To a solution of compoundYL-015-10 (40 mg, 0.054 mmol) and N-hydroxysuccinimide (13 mg, 0.115mmol) in anhydrous DMF (3 mL), was added EDC (31 mg, 0.162 mmol). Thesolution was stirred at room temperature for 24 hours. DMF was removedby vacuum distillation and the residue was further dried on high vacuum.The residue was then dissolved in a minimum amount of MeOH (0.12 mL) andH₂ O (3.2 mL) was added to induce precipitation. The precipitates werewashed with H₂ O (3×0.8 mL) and dried on vacuum to give a solid product(31 mg) (Yield: 69%). ¹ HNMR (MeOH-d4, 300 MHz).

8. Synthesis of Argatroban-C6 NHS Ester (compound YL-015-35).

To a solution of compound YL-015-15 (78 mg, 0.12 mmol) andN-hydroxysuccinimide (29 mg, 0.25 mmol) in anhydrous DMF (6 mL) wasadded EDC (72 mg, 0.38 mmol). The solution was stirred at roomtemperature for 20 hours and DMF was removed by vacuum distillation. Theresidue was dissolved in a minimum amount of MeOH (0.4 mL) and H₂ O (1.2mL) was added to induce precipitation. The precipitates were washed withH₂ O (3×0.7 mL) and dried on vacuum to afford a solid product. The solidproduct was further purified by recrystallization from acetone/ether(1/1, v/v) to give a white solid product (62 mg) (Yield: 69%). ¹ HNMR(MeOH-d4, 300 MHz).

9. Synthesis of Argatroban-C12 Sulfo-NHS Ester (compound YL-015-23).

Compound YL-015-10 (10.0 mg, 13.5 μmol), sulfo-NHS (6.2 mg, 28.3 μmol)and EDC (8.2 mg, 42.5 μmol) were dissolved in anhydrous DMF (0.8 mL).The reaction mixture was stirred at room temperature for 2 days and DMFwas removed by vacuum distillation. The residue was washed with a smallamount of H₂ O, then with EtOAc and acetone, to give a white solidproduct (9.2 mg) (Yield: 77%). ¹ HNMR (MeOH-d4, 300 MHz).

Results: A one-step derivatization of the thrombin inhibitor argatrobanwith a linking polypeptide and a chemically reactive group has beenrealized. Briefly, the free carboxylate group on the argatroban moleculeis activated with HBTU, presumably via formation ofN-hydroxybenzotriazole ester, which then reacts with the amino group ofthe linking polypeptide to give the desired adduct. A side-product witha molecular weight corresponding to that of argatroban minus a watermolecule, was also observed by mass spectroscopic analysis. Thisby-product is probably derived from an internal nucleophilic attack onthe activated ester. If the argatroban molecule was pre-activated withHBTU before the addition of the nucleophilic amino ester, more of theside product would be produced. Therefore, the amino ester andargatroban were mixed prior to addition of HBTU in the reaction. Theproduct was purified on preparative TLC plates. Although the yields arerelatively low (28-53%), the simple one-step linking polypeptideplacement without protecting those functional groups on argatroban isvery satisfactory.

Two amino aliphatic acid methyl esters with 6 and 12 methylene unitsspans were chosen as the linking polypeptides because of theirstructural stability and simplicity. The linking polypeptides ofvariable lengths will provide information on length requirement foroptimal drug presentation to its target.

Subsequent hydrolysis of the methyl esters on the linking polypeptidesunder alkaline condition went smoothly to give the corresponding acid in73-85% yields. Treatment of the free acid with HBTU in the presence ofN-hydroxysuccinimide and DIEA afforded the desired NHS ester in 42%yield. The yield was later improved to 69% by using EDC as the couplingreagent. The formation of the NHS ester was confirmed by ¹ HNMR and massspectroscopy.

Example 2 Derivatized Argatroban Molecules are Bioavailable afterCovalent Attachment to Rabbit Erythrocytes

Material and Methods: Rabbit erythrocytes were conjugated with theArgatroban-C6 NHS ester compound (compound YL-015-35). To do so, rabbiterythrocytes were isolated from fresh, citrated blood. In brief, analiquot of blood was centrifuged for 5 minutes at 1800 rpm (SorvallRC-5B, SH-3000 rotor). The buffy coat was pipetted off the top layer ofthe cell pellet and the remaining erythrocytes were washed four timeswith PBS, pH 7.4. Before the last wash, an aliquot of cells was removedfor counting. The washed erythrocytes were resuspended at a density of1×10⁹ cells/mL. The total volume of cells labeled per sample was 3 mL.Cells were reacted at room temperature for 45 minutes with gentleagitation with one of the following reagents: (1) sham, 0.5% DMSO/PBS;(2) 100 μM argatroban C6-free acid (compound YL-015-15) in 0.5%DMSO/PBS; (3) 10 μM argatroban C6-NHS ester (compound YL-015-35) in 0.5%DMSO/PBS; (4) 50 μM argatroban C6-NHS ester (compound YL-015-35) in 0.5%DMSO/PBS; (5) 100 μM argatroban C6-NHS ester (compound YL-015-35) in0.5% DMSO/PBS; and (6) 100 μM sulfo-NHS-LC-biotin (Pierce, catalog no.21335) in 0.5% DMSO/PBS. After the labeling reaction, the cells werecentrifuged 4 minutes at 1800 rpm and the supernatant was discarded. Thecells were resuspended in 10 ml of 0.5% DMSO/PBS and pelleted. The cellswere washed one more time with 10 ml of PBS.

To quench any remaining reactive esters, the cells were then resuspendedin 6 mg/mL BSA/PBS (Sigma, A-2934, lot 93HO291) and gently agitated atroom temperature for 30 minutes. After quenching, the cells were washedfive times with PBS. Cell yields were determined by cell counting.

After conjugation, the cells were hypotonically lysed and the ensuingmembrane (ghost) and cytosol fractions were assayed by immunoblottingand ELISA. Specifically, conjugated eiythrocyte pellets were chilled onice for 5 minutes. Cells were then lysed on ice with 12 ml of ice-cold 5mM phosphate buffer, pH 8.0 (PB) containing 1 mM Pefabloc and 40 μMleupeptin. Lysates were briefly vortexed and then centrifuged for 20minutes at 4° C. at 15,000 rpm (SS-34, Sorvall RC-5B). The supernatant(hemolysate) was pipetted off the pellet, labeled and stored at 40° C.The pellets (ghosts) were washed four times with 12 ml of cold PBS andcentrifuged as above. The protein content of the ghosts and hemolysateswas determined by using the Micro BCA kit (Pierce; catalog no. 23231).Cell fractions were stored at -80° C.

Samples were then solubilized in 2× reducing cocktail (5% SDS, 50%glycerol, 0.5M 2-mercaptoethanol in 0.25M Tris-HCI, pH 6.8). After a 5minute treatment at 100° C. and then brief cooling, samples wereresolved by SDS-PAGE on 10% polyacrylamide mini-gels. Afterelectrophoretic transfer to nitrocellulose and brief Ponceau S staining,the blots were blocked with Blotto (5% instant non-fat dry milk in PBS,pH 7.4) for 2 hours at room temperature. The blots were then washed fourtimes with TBS-T (20 mM Tris-HCI, pH 7.6, 137 mM NaCl, 0.25% Tween-20)and incubated for 2 hours at room temperature with a 1:1000 dilution ofrabbit serum in TBS-T. Blots were then washed four times with TBS-T asabove and incubated with biotinylated anti-rabbit IgG antibodies(Vector, BA-1000) for 1 hour at room temperature. Blots were washed asabove and then were incubated with ABC--HRP (Vector) for 30 minutes atroom temperature. After washing, blots were visualized with the metalenhanced DAB substrate kit (Pierce, catalog no. 34065).

Results: The results of the above described experiments demonstratedthat numerous erythrocyte membrane proteins were covalently modifiedwith the derivatized argatroban-C6 NHS ester molecule. By contrast, thepresence of derivatized argatroban molecules on cytosolic proteins wasat the limit of detection, suggesting that this thrombin inhibitorcompound is an effective reagent for covalently bonding to proteinslocated on the outside of cells and, ultimately, for the delivery ofdrugs. Moreover, by a capture ELISA assay, it was observed that aportion of the thrombin inhibitor-labeled proteins belong to theglycophorin family of proteins.

Example 3 Mass Spectrometric Analysis of Derivatized ArgatrobanConjugation to Human Serum Albumin (HSA) and Rabbit Serum Albumin (RSA)

Materials and Methods: Mass spectrometry was used to support covalencyof the bonding of derivatized argatroban molecules to the proteins humanserum albumin (HSA) and rabbit serum albumin (RSA) as well as toquantitate the number of conjugated argatroban molecules covalentlybound to the proteins. All samples were analyzed using a highperformance liquid chromatograph (HPLC) coupled in-line to anelectrospray ionization (ESI) mass spectrometer (MS) which is termedHPLC/ESIMS. Some samples were also analyzed using a matrix-assistedlaser desorption ionization (MALDI) time-of-flight (TOF) massspectrometer which is termed (MALDI-TOFMS).

Samples for HPLC/ESIMS were either prepared as duplicate reactionsamples or were part of the same reaction samples which were used in theELISA assays described below. These samples were injected directly onlythe HPLC/ESIMS without any manipulation other than dilution with water,where the PBS, DMSO, and unreacted derivative molecules are separatedfrom the protein-argatroban derivative molecule conjugate. Samples forMALDI-MS were first HPLC-purified and mixed 1:1 directly on thestainless steel analysis plate with a matrix of 10 mg/ml sinnapinic acidin 50% acetonitrile/50% water. The mixture was then dried with a gentleflow of air to allow for crystallization and inserted into theinstrument for laser ionization.

The HPLC is a Hewlett Packard 1090 Series II liquid chromatogram with adiode array detector. The column used for separation is a Brownleeaquapore OD-300 reversed phase C18 column, 1 mm×10 cm, 300 Å pore size,7 μm particle diameter. The mass spectrometer to which the HPLC isconnected using fused Silica capillary tubing is a Sciex API-300 triplequadrupole mass spectrometer with an ion-spray source to allow foratmospheric pressure ionization of a liquid flowing continuously intothe instrument. The MALDI-TOFMS is a Perceptive Biosystems Voyager EliteDE instrument run in the linear mode.

Results:

(1) Argatroban-C6 NHS Ester vs. Argatroban-C12 NHS Ester. 20:1 MolarExcess of Ester vs. HSA or RSA.

HPLC/ESIMS of HSA (0.33 μg/μl, 5 μM) reacted for 1 hour at roomtemperature with 100 μM argatroban-C6 NHS ester (compound YL-015-35)indicated a covalent addition of 1-8 argatroban derivative molecules perHSA molecule. RSA (0.30 μg/μl, 4.5 μM) reacted for 1 hour at roomtemperature with 100 μM argatroban-C6 NHS ester (compound YL-015-35)also indicated a covalent addition of 1-8 argatroban derivativemolecules per RSA molecule. However, with the argatroban-C12 NHS ester(compound YL-015-11) under the same conditions, only 1-5 argatrobanderivatives are covalently added per molecule of HSA or RSA. Theseresults suggest that the argatroban derivative molecule having a linkingpolypeptide of 6 carbon atoms acts to provide more efficient bonding toavailable reactive finctionalities on both HSA and RSA than does anargatroban derivative molecule having a linking polypeptide of 12 carbonatoms.

(2) HSA vs. RSA. Argatroban-C12 NHS Ester. 1:10 Molar Deficiency ofEster vs. Protein

HPLC/ESIMS of HSA (0.33% μg/μl, 5 μM) reacted for 1 hour at roomtemperature with 0.5 μM argatroban-C12 NHS ester (compound YL-015-11)indicated a covalent addition of 0-4 argatroban derivative molecules perHSA molecule. RSA (0.30 μg/μl, 4.5 μM) reacted for 1 hour at roomtemperature with 0.5 μM argatroban-C12 NHS ester (compound YL-015-11)indicated a covalent addition of 0-3 argatroban derivative molecules perRSA molecule, or slightly less covalent labeling than obtained with theHSA protein.

(3) 15 μM vs. 30 μM RSA. Argatroban-C6 NHS Ester, 10-12:1 Molar Excessof Ester vs. RSA

HPLC/ESIMS of RSA (1.0 μg/μl, 15 μM) reacted for 1 hour at roomtemperature with 200 μM argatroban-C6 NHS ester (compound YL-015-35)indicated a covalent addition of 1-9 argatroban derivative molecules perRSA molecule. RSA (2.0 μg/μl, 30 μM) reacted for 1 hour at roomtemperature with 300 μM argatroban-C6 NHS ester (compound YL-015-35)also indicated a covalent addition of 1-9 argatroban derivativemolecules per RSA molecule. These results were confirmed by MALDI-MSanalysis of these two samples which gives a single broad peak centeredon the average molecular weight of the sample. Based on these averagemolecular weights, the 15 μM/200 μM RSA/argatroban derivative sampleyielded an average of 5.6 argatroban derivative molecules added per RSAmolecule (range of 1-10 added) and the 30 μM/300 μM RSA/argatrobanderivative sample yielded an average of 5.4 argatroban derivativemolecules added per RSA molecule (range of 1-10 added).

(4) Concentration Study of 1-100 μM Argatroban-C6 NHS Ester Reacted with4.5 μM RSA

HPLC/ESIMS of RSA (0.30 μg/μl, 4.5 μM) reacted for 1 hour at roomtemperature with argatroban-C6 NHS ester (compound YL-015-35) (1, 10,15, 20, 25, and 100 μM) yielded a linear covalent addition of argatrobanderivative molecules to each molecule of RSA as indicated by thefollowing results:

1 μM adds no detectable argatroban derivative molecules

10 μM adds 1 argatroban derivative molecule to ⁻ 50% of the RSAmolecules

15 μM adds 0-3 argatroban derivative molecules per RSA molecule(majority of RSA molecules are +1)

20 μM adds 1-3 argatroban derivative molecules per RSA molecule(majority of RSA molecules are +1 and +2)

25 μM adds 1-4 argatroban derivative molecules per RSA molecule(majority of RSA molecules are +1 and +2)

100 μM adds 2-10 argatroban derivative molecules per RSA molecule (nomajor species).

Example 4 Sandwich ELISA Assays to Assess the Relative Bioavailabilityof Derivatized HSA to Thrombin

Materials and Methods: Human serum albumin (HSA, Sigma Cat. #A-8763) ata concentration of 333 μg/ml or rabbit serum albumin (RSA, Sigma Cat#A-9438) at a concentration of 300 μg/ml was reacted with 100 μMargatroban-C6 or -C12 NHS ester (compounds YL-015-35 or YL-015-11,respectively) in PBS, pH 7.4, for 1 hour at room temperature. Thereaction was quenched by the addition of hydroxylamine, pH 7.8 to afinal concentration of 50 mM and incubation at room temperature for 10minutes. Prequenched negative control samples were of the samecomposition except that the argatroban NHS ester was reacted withhydroxylamine for 10 minutes prior to the addition of HSA or RSA.Finally, the HSA or RSA only sample had DMSO added to 1% to mimic theaddition of argatroban NHS ester solutions and was incubated andquenched the same as the other reactions.

Rabbit anti-HSA (Boehringer/Mannheim, catalog no. 605001) or goatanti-RSA (Cappel, catalog no. 55629) antibodies were diluted 1:5000 inPBS, pH 7.4 and 100 μl/well was aliquotted into a NUNC Maxisorp F96plates (catalog no. 439454). The plate was incubated overnight at 4° C.,then blocked by the addition of 200 μl/well of 1% BSA/PBS and incubatedat room temperature for 1 hour. The plate was then washed 5 times bysubmersion in PBS, pH 7.4.

The various HSA/argatroban derivative or RSA/argatroban derivativesamples were serially diluted in 1% BSA/PBS (1:100 to 1:50,000) and 100μl/well was added to duplicate wells of each sample dilution. Sampleswere incubated on the plates for 2 hours at room temperature and theplates were then washed as above.

Thrombin (Via Enzymes Systems, 11.4 mg/ml) was diluted to 9.4 μg/ml inPBS, pH 7.4, with 0.1% BSA/0.1% PE-G 8000 (Sigma, catalog no. P-2139)and 100 μl/well was added. The plates were incubated at room temperaturefor 1 hour and washed as above.

Mouse anti-thrombin (American Diagnostics, catalog no. EST-7), anantibody thought to bind away from the active site of thrombin, wasdiluted to 1 μg/ml in PBS, pH 7.4, with 0.1% BSA/0.1% PEG 8000 and 100μ/well was added to all wells. The plates were incubated at roomtemperature for 30 minutes and washed as above.

Biotinylated rabbit anti-mouse IgG (mouse gamma specific, Zymed, catalogno. 61-6540) was diluted 1:1000 in PBS, pH 7.4, with 0.1% BSA and 1% PEG8000 and 100 μl/well was added to all wells. The plates were incubatedat room temperature for 30 minutes and washed as above.

Next, 100 μl/well of ABC-HRP (Vector, catalog no. PK4000) was added,each component diluted 1:500 in PBS, pH 7.4, with 0.1% TWEEN 20 andincubated 30 minutes at room temperature prior to use. PEG 8000 wasadded to 0.1% just before addition of ABC-HRP to the plates. The plateswere incubated with ABC--HRP for 30 minutes at room temperature and werethen washed 10 times by submersion in PBS, pH 7.4.

Alternatively to adding biotinylated rabbit anti-mouse IgG and theABC--HRP, the assay was simplified and background reduced by adding goatanti-mouse IgG conjugated with HRP (Jackson ImmunoResearch, catalog no.115-035-146) diluted 1:500 in HRP diluent (Medix, catalog no. RIH4203)and 100 μl/well added. The plates were incubated at room temperature for30 minutes and were then washed 10 times by submersion in PBS, pH 7.4.

Finally, 100 μl/well of o-phenylenediamine dihydrochloride (OPD, Sigma,catalog no. P-3804) was added at 0.5 mg/mi in citrate-phosphate buffer,pH 5.3 with 0.015% H₂ O₂ and incubated at room temperature for 30minutes. After incubation, 200 μl/well of 2N sulfuric acid (VWR, catalogno. VW3S00-1) was added and absorbance was read at OD₄₉₀ in a SpectraMaxplate reader (Molecular Devices) after agitating the plate for 5seconds.

Results: The results obtained from the above sandwich ELISA assays arepresented in FIGS. 2, 3 and 4. The data presented in FIG. 2 demonstratesthat there is a significant increase of OD₄₉₀ in wells incubated withsamples containing HSA reacted with argatroban-C6 or -C12 NHS ester(compounds YL-015-35 and YL-015-11, respectively) as compared to samplescontaining prequenched argatroban NHS ester or HSA alone. A significantsignal above background was detected at approximately 10 ng/ml for boththe C6 and the C12 derivatized compounds but the absolute signals forthe two compounds are very different with the C6 having a significantlyhigher OD (approx 6-7 fold at saturation). These data suggest that theargatroban NHS ester derivative molecules have covalently bound to theHSA protein in such a way that it is able to interact and bind withthrombin which, in turn, can then be detected by an anti-thrombinantibody. These data, therefore, suggest that the argatroban derivativescovalently bound to the HSA protein is capable of binding to andinhibiting thrombin.

In the case of RSA, only the argatroban-C6 NHS ester derivative has beendetected in an ELISA format (see FIG. 3). A significant signal abovebackground was detected at approximately 30 ng/ml for the argatroban-C6derivative. In this assay, the absolute signal is lower than thatobtained with HSA. This maybe the result of differences in the assay dueto the capture antibody, to differences in the extent and availabilityof labeling, or both.

Covalency of the bond between the argatroban NHS ester and the HSA orRSA protein was supported by mass spectrometry data (obtained from theduplicate reaction samples or the rest of the same reaction sample,described above) that showed that the argatroban derivative moleculeshad created covalent adducts on the majority of the HSA or RSA proteinspresent in the samples.

Human glycophorin A protein (hGPA) has also been reacted with theargatroban-C6 NHS ester derivative (compound YL-015-35) under similarconditions as for the RSA protein described above. The results of thesestudies are presented in FIG. 4. As is shown in FIG. 4, the hGPA proteinreacted with the argatroban-C6 NHS derivative and was shown by ELISA tobind thrombin to give a detectable signal at approximately 30 ng/ml. TheELISA assay was identical to the one described for HSA and RSA abovewith the exception that mouse anti-hGPA (BioAtlantic/CRTS, mAb 5F4),diluted to 10 μg/ml in 0.1M sodium acetate, pH 4.5, was used to capturethe NHS-argatroban derivative reacted hGPA protein.

The results of the above experiments clearly demonstrate that theargatroban derivative molecules of the present invention not onlycovalently bond to reactive functionalities on variousvascular-associated proteins, but do so in a way not to inhibit theability of the derivative molecule to bind to and inhibit thrombin.

Keyhole limpet cyanin (KLH, Sigma, catalog no. R-5755) has also beenreacted with argatroban-C12 NHS ester (compound YL-015-11) under similarconditions as for the HSA protein described above. The KLH proteinreacted with argatroban-C12 NHS ester (compound YL-015-11) was shown byELISA assay to bind to thrombin and give a detectable signal atapproximately 100 ng/ml. The ELISA assay was identical to the onedescribed above for HSA and RSA above with the exception that rabbitanti-KLH (Cappel, catalog no. 55966) diluted 1:5000 in PBS, pH 7.4 wasused to capture the NHS-argatroban derivative reacted KLH. Because ofthe extremely heterogeneous nature of the KLH preparation, it was notpossible to analyze these samples by mass spectrometry. However, similarmaterial (reaction containing KLH at 2 mg/ml and argatroban-C12 NHSester at 3.3 μM) was used to generate polyclonal antibodies to theargatroban derivative, thereby confirming the covalent modification ofthe KLH protein.

Example 5 Sandwich ELISA Assays Using Polyclonal Antibodies to EvaluateHSA. hGPA and Rabbit Erythrocyte Ghosts Conjugated with Argatroban-C6NHS Ester

Materials and Methods: Human serum albumin (HSA, Sigma catalog no.A-8763) or human glycophorin A (HGPA) protein at a concentration of 300μg/ml was reacted with 100 μM argatroban-C6 NHS ester (compoundYL-015-35) in PBS, pH 7.4, for 1 hour at room temperature. The reactionwas quenched by adding hydroxylamine, pH 7.8 to a final concentration of50 mM and incubating at room temperature for 10 minutes. Prequenchednegative control samples were of the same composition except that theargatroban-C6 NHS ester derivative was reacted with hydroxylamine for 10minutes prior to the addition of HSA or hGPA protein. Finally, the HSAor hGPA only samples had DMSO added to 1% to mimic addition ofargatroban derivative solutions and was incubated and quenched the sameas other reactions.

Rabbit anti-HSA monoclonal antibody (Boehringer/Mannheim, catalog no.605001) diluted 1:5000 in PBS, pH 7.4 or mouse anti-hGPA monoclonalantibody (BioAtlantic/CRTS, mAb 5F4, diluted to 10 μg/ml in 0.1M sodiumacetate, pH 4.5 or mAb 3C10 diluted 1:500 or mAb 3E4 diluted 1:1000 inPBS, pH 7.4--each antibody recognizes a different epitope of hGPA), wereused to capture respective proteins by aliquotting 100 μl/well into aNUNC Maxisorp F96 plate (catalog no. 439454). The plates were incubatedovernight at 4° C., were then blocked by the addition of 200 μl/well of1% BSA/PBS and incubated at room temperature for 1 hour. The plates werethen washed 5 times by submersion in PBS, pH 7.4.

The various HSA/argatroban derivative or hGPA/argatroban derivativesamples were serially diluted in 1% BSA/PBS (1:100 to 1:50,000) and 100μl/well was added to duplicate wells for each sample dilution. Sampleswere incubated on the plates for 2 hours at room temperature and thenwere washed as above.

Rabbit anti-KLH-argatroban antibody was diluted 1:500 or 1:1000 in 1%BSA/PBS, pH 7.4 and 100 μl/well added to each well. The plates were thenincubated at room temperature for 30 minutes and washed as above.

Biotinylated goat anti-rabbit IgG (Vector, catalog no. BA1000) wasdiluted 1:1000 in 1% BSA/PBS, pH 7.4 and 100 μl/well added to all wells.The plates were incubated at room temperature for 30 minutes and washedas above.

Next, 100 μl/well of ABC-HRP (Vector, catalog no. PK4000) was added,each component diluted 1:500 in PBS, pH 7.4, with 0.1% TWEEN 20 andincubated 30 minutes at room temperature prior to use. The plates werethen washed 10 times by submersion in PBS, pH 7.4.

Finally, 100 μl/well of o-phenylenediamine dihydrochloride (OPD, Sigma,catalog no. P-3804) was added at 0.5 mg/ml in citrate-phosphate buffer,pH 5.3 with 0.015% H₂ O₂ and incubated at room temperature for 15-20minutes. Then, 100 μl/well of 2N sulfuric acid (VWR, catalog no.VW3500-1) was added and absorbance read at OD₄₉₀ in a SpectraMax platereader (Molecular Devices) after agitating the plate for 5 seconds.

Results: The results of the above described experiments are presented inFIGS. 5, 6, 7 and 8. Specifically, there was a significant increase ofOD₄₉₀ in wells incubated with samples reacted with argatroban-C6 NHSester (compound YL-015-35) as compared to samples containing prequenchedargatroban-C6 NHS ester or protein alone. A significant signal abovebackground was detected at approximately 13 ng/ml for HSA-argatrobanderivative (see FIG. 5) and 12 ng/ml for hGPA-argatroban derivative (5F4mAb, see FIG. 6). The other anti-hGPA monoclonal antibodies (3C10 and3E4) also detected hGPA-argatroban derivatives, however, at lower levels(see FIGS. 7 and 8, respectively). These data confirm thethrombin/anti-thrombin results showing that the argatroban-C6 NHS esterhas covalently bound to the HSA and hGPA proteins and, once bound, bindsto thrombin.

Rabbit erythrocyte ghosts that had been prepared and reacted with 100 μMargatroban-C6 NHS ester (compound YL-015-35) were also shown to bepositive in an plate ELISA format using the polyclonalanti-KLH-argatroban antibodies to detect (rabbit erythrocytes reactedwith 50 μM argatroban-C6 NHS ester were also tested but the signal wasmuch lower). Sham treated rabbit erythrocytes or rabbit erythrocytestreated with the argatroban-C6 free acid (compound YL-015-15) did notgive a signal above background. The ELISA assay was similar to the onedescribed above for the HSA and RSA proteins with the exception thateither goat anti-rabbit erythrocyte (Cappel, catalog no. 55616) diluted1:500 or mouse anti-hGPA (BioAtlantic, mAb 3C10) diluted 1:100 in PBS,pH 7.4 were used to capture the argatroban-C6 NHS ester reacted rabbiterythrocyte ghosts. The argatroban-C6 NHS ester was then detected usingthe polyclonal anti-KLH-argatroban antibodies (1:1000) in the samemanner as for the HSA and hGPA proteins above. As a positive control,hGPA labeled with argatroban-C6 NHS ester (same material as above) wasincluded in both assays and detected by the goat anti-rabbit erythrocyteto 60 ng/ml and the mouse anti-hGPA to 30 ng/ml. Using the goatanti-rabbit erythrocyte to capture, rabbit erythrocyte/NHS-argatrobanconjugate was detected to 1.6-3.2 μg/ml of total protein. Using themouse anti-hGPA (mAb 3C10) to capture, rabbit erythrocyte/NHS-argatrobanconjugate was detected at about 160 μg/ml of total protein.

These results indicate that the argatroban-C6 NHS ester derivative hascovalently attached to the rabbit erythrocytes in a manner that isdetectable using polyclonal anti-KLH-argatroban antibodies. Since themouse anti-hGPA was successful with rabbit cells, this assay should alsobe able to detect argatroban-C6 NHS ester that has covalently attachedto human erythrocyte ghosts and whole cells. Moreover, because the goatanti-rabbit erythrocyte antibody was able to detect hGPA, it is likelythat this reagent could be used for human erythrocyte ghosts and cellsas well.

Example 6 Detection of Thrombin Inhibitory Activity

A variety of assays were employed to detect the ability of argatrobanderivatives to inhibit thrombin activity. Specifically, a Km-basedchromogenic assay was first employed. Initially, a 1 mL total volumecontaining argatroban or a derivative thereof, 8 μM S-2238(H-D-Phe-Pip-Arg-p-nitroaniline2 HCl) as a substrate forthrombin-induced hydrolysis in 0.1% PEG, 1% DMSO in PBS, pH 7.2, 35° C.was initiated by the addition of 10 mU of thrombin from human plasma andthe absorbance was measured for 10 minutes at 405 nm. Measurements at405 nm are designed to detect the thrombin-induced liberation ofp-nitroaniline from the S-2238 substrate. As such, an observed increasein absorbance at 405 nm directly correlates with an increase inhydrolysis of the substrate by thrombin. The results from these studiesindicated that the IC₅₀ for underivatized argatroban was 20 nM, whereasthe IC₅₀ for a C12-tethered carboxylate of argatroban was 150 nM.

Similar results were obtained in a Km-based 96-well formattedfluorogenic assay employing 25 μMN-t-Boc-β-Benzyl-Asp-Pro-Arg-7-Amido-4-MethylCoumarin HCI as ahydrolysis substrate (total volume 250 μl, 25 μM substrate, in 0.1 mg/mlBSA, PBS in 1% DMSO initiated by addition of 2.5 mU of thrombin fromhuman plasma and absorbance measured for 10 minutes at 460 nm). Formthese studies, the IC₅₀ for underivatized argatroban was 6 nM, for aC12-tethered carboxylate of argatroban, about 80 nM, and for compoundYL-015-15, a C6-tethered carboxylate of argatroban, about 50 nM.

In a Vmax-based assay, (total volume 250 μl, 200 μM S-2238 as hydrolysissubstrate in 0.1% PEG, 1% DMSO in PBS, pH 7.2, 25° C. initiated with 40mU of thrombin from human plasma and measured for 20 minutes at 405 nm)the IC₅₀ for underivatized argatroban was 250 nM. The IC₅₀ for aC12-tethered carboxylate of argatroban in this assay was about 5-8 uM.Argatroban derivatized with a C6 instead of a C12-tether had an lC₅₀ of1.25 to 10 uM, depending on whether in free acid or methyl ester form.

Reaction conditions for conjugation of HSA with an NHS ester derivativeof argatroban: 5 μM HSA was incubated with 235 μM of NHS ester ofargatroban in 1.5 DMSO, 1.5% ethanol in PBS, pH 7.4 for 1.5 hours atroom temperature. At this point, the unreacted ester in the sample wasquenched by addition of 1.2 mM hydroxylamine and held at roomtemperature for 30 minutes. Conjugated HSA was desalted on a kwiksepcolumn to remove any unbound forms of the argatroban derivative andconcentrated using a centricon-10 column to approximately the initialreaction volume. Appropriate analogous procedures were followed forcontrols using 1) underivatized HSA protein and 2) HSA protein processedwith prequenched argatroban NHS ester.

In a Km-based inhibition assay for thrombin from human plasma, 2.5 mU ofthrombin in 10 μl of 0.1% polyethylene glycol 8000 (PEG) in PBS, pH 7.2, were added to 240 μl of PBS containing as final concentrations 10 μMS-2238 hydrolysis substrate, 1% DMSO, 0.1% PEG, and with or without 40μg/ml HSA. HSA proteins tested included HSA conjugated with an NHS esterof argatroban, HSA processed with hydroxylamine prequenched NHS ester ofargatroban and HSA processed through the same incubations and desaltingsused to remove residual or free acid forms of the NHS ester ofargatroban as in the above samples. The rate of substrate hydrolysis bythrombin was nearly linear for the first ten minutes in all cases, asmeasured by an increase in absorbance at 405 nm from the production offree p-nitroaniline. In duplicate wells 1) lacking HSA or 2) containingunderivatized HSA or 3) containing HSA processed with quenched NHS esterof argatroban, the rate of hydrolysis of substrate was indistinguishablebetween samples and ranged from 0.94 to 1.08 mOD/minute. In duplicatewells containing HSA conjugated with the NHS ester of argatroban, therate of hydrolysis of substrate ranged from 0.66 to 0.71 mOD/minute,thus, 32% inhibited compared to controls.

In a second independent experiment, HSA was again conjugated withargatroban-C12 NHS ester (compound YL-015-11). Controls containing HSAwith prequenched NHS ester, or HSA alone, were also prepared. Sampleswere assayed in duplicate, and a concentration range of HSA of 20, 40,and 80 mg/ml tested. HSA conjugated with argatroban-C12 NHS esterinhibited thrombin activity from 35-42% at 80 mg/ml compared to controlsamples, with 15-20% inhibition at 40 mg/ml relative to control samples.

Example 7 K_(m) -Based Fluorogenic Assay for Inhibition of Thrombin fromHuman Plasma

Thrombin from human plasma was obtained from Enzyme System Products,Dublin, Calif., in glycerol:water 1:1, 11.4 mg/ml, 3800 U/mg, MW 36,700,and stored undiluted at -20° C. It was freshly diluted the day of use.

To dilute for the assay, 0.67 μl (25 Units) of thrombin was pipettedinto 5 ml of 0.1 mg/ml BSA (from Pierce, Catalog No. 23209, BSA fractionV, a 2 mg/ml stock in saline with sodium azide), in cold PBS (GIBCO,Catalog No. 14190-136, without calcium chloride and magnesium chloride,pH 7.2) and briefly vortexed, yielding a stock of 5 mU thrombin/μl. Asecond dilution of 250 μl of this stock into 9.75 ml of 0.1 mg BSA/mlPBS resulted in a 0.125 mU/μl, or 3.125 mU/25 μl, final working stock.

The substrate was N-tBOC-β-Benzyl-Asp-Pro-Arg-7-Amido-4-MethylcoumarinHCl and was obtained from Sigma, Catalog No. B-4028, Lot 122H0070, F.W.770.3. Five mg was dissolved in 1.42 ml DMSO for a 4 mM stock. The stockwas stored in 200 μl aliquots at -20° C. in the dark.

An alternative substrate was N-tBOC-Val-Pro-Arg-7-Amido-4-MethylcoumarinHCl and was obtained from Sigma, Catalog No. B-9385, Lot 53H0805, F.W.of free base 627.7. Five mg was dissolved in 1.863 ml DMSO for a 4 mMstock. The stock was stored in 200 μl aliquots at -20° C. in the dark.

The fluorescent standard was 7-Amido-4-Methylcoumarin and was obtainedfrom Aldrich, Catalog No. 25,737-0, MW 175.19. 6.1 mg was dissolved in1.743 ml of DMSO for a 20 mM stock. The stock was stored in 200 μlaliquots at -20° C. in the dark.

Assays were performed kinetically at ambient temperature (about 20° C.)in 96-well microtiter plates (Nunc Immunomodule Maxisorb, a flatbottomed polystyrene plate) on a Perseptive Biosystems Cytofluor I.Stock solutions containing common concentrations and components for eachwell or group of well were prepared and 200 μl aliquotted per microtiterwell. Assays were routinely conducted in a total volume of 250 μl, whichallows for 25 μl for inhibitor and 25 μl for thrombin (about 3 mU).

For an initial determination of K_(m) and V_(max), the concentration ofthe fluorogenic substrateN-tBOC-β-Benzyl-Asp-Pro-Arg-7-Amido-4-Methylcoumarin was varied from 5to 200 μM.

For inhibition assays, final concentrations per well (250 μl V_(T))included 25 μM N-tBOC-β-Benzyl-Asp-Pro-Arg-7-Amido-4-Methylcoumarin HCl(approximately K_(m)), 0.1 mg BSA/ml PBS, and 1% DMSO. Initial readingswere taken to ensure exclusion of rogue wells with abnormally highbackgrounds (λ excitation at 360 nM, bandwidth 40 nM, λ emission, 460nm, bandwidth 40 nm). Then, 25 μl of a 10× stock of the putativeinhibitor were added per well, mixed for 5 seconds and read kineticallyfor 10 minutes at ambient temperature to assess background interferenceand/or any increase in hydrolysis of substrate due to inhibitor.

In inhibition assays using argatroban or soluble, tethered variants ofargatroban (C6 or C12), samples were dissolved in DMSO at 1 mM, thendiluted appropriately for assay in 1% DMSO in the buffer specifiedabove.

Derivatized argatroban molecules attached to RSA or ghosts derived fromred blood cells by an activator were formulated in phosphate buffer.Additional RSA of ghost samples processed (1) with tethered argatrobanmolecules lacking activator or (2) with activator groups attached toligands that do not inhibit thrombin were included as negative controls.

The thrombin assay was then initiated by adding 25 μl of thrombin/wellwith a multichannel pipettor and mixed for 5 seconds. The appearence ofthe fluorescent product (free 7-amido-4-methylcoumarin released from thepeptide by thrombin) was followed simultaneously in 48 wells (onereading per minute for 30 minutes, λ excitation at 360 nm, bandwidth 40nm, λ emission, 460 nm, bandwidth 40 nm). The velocity of the reactionwas deterined as an initial velocity in the first 10 minutes, with thesignal in relative fluorescent units (RFU) of an uninhibited reactionabout 9,000-10,000, above an initial background reading of about 1,000(gain setting of 80). Routinely, less than 5% of the initial substratewas converted to fluorescent product during 10 minutes, and samples andcurves are run in duplicate. A standard curve of thrombin activity from0.3125-3.5 mU/well was included, as well as a standard curve of7-amido-4-methylcoumarin (ranging from 5 to 200 pmoles/well in 1% DMSO,0.1 mg BSA/ml PBS, V_(T) 250 μl/well). The pH of each sample wasverified as 7.2 with pH paper at the end of the assay.

The results of the above assays are shown in FIGS. 9 and 10.Specifically, FIG. 9 illustrates the ability of the RSA-argatroban (C6)conjugate to inhibit thrombin. The lC₅₀ for this conjugate was estimatedto be 25 nM. By contrast, the control RSA sample was not inhibitory atany concentration assayed. This result confirms that the C6-tetheredargatroban molecules which are covalently-linked to the RSA protein arebioactive as well as bioavailable. Indeed, this observation was extendedto erythrocyte ghosts which were labeled with the C6-tethered argarobanmolecule (see FIG. 10). This graph illustrates a kinetic assay ofincreasing aliquots of ghosts derived from erythrocytes labeled with 100μM argatroban (C6)-NHS ester. Control sham-treated ghosts or ghostsderived from cells treated with the corresponding argatrobanC6-carboxylate were not inhibitory (data not shown). Taken together,these data demonstrate that carrier proteins, either cellularly- ornon-cellularly-associated, when conjugated to tethered argatrobanmolecules are effective drug carriers and that this treatment maintainsthe bioactivity of the thrombin inhibitor.

Example 8 Platelet Aggregation Assays

A thrombin-induced platelet aggregation assay was selected to furtherconfirm the bioactivity of the tethered argatroban (C6)-RSA conjugates.The conjugate was prepared according to the following protocol. A 15 μMsolution of RSA in PBS was reacted with 200 μM argatroban-C6 NHS esterfor 1 hour at room temperature. Sham samples consist of the RSAincubated for the same length of time at RT in 2% DMSO in PBS. Each ofthe samples was then concentrated to a final volume of 1.5 mL usingAmicon Centricon 30 filtration/concentration devices. The concentratedsamples were then desalted on Pierce's 5 mL Kwiksep excellulose plasticdesalting columns. The excluded volumes containing RSA orargatroban-C6-RSA were then subjected to BCA microprotein estimation andlyophilization. In addition, these samples were analyzed by LC/MS,sandwich ELISA assays (described above) to measure thrombin binding andimmunoblotting. From LC/MS analysis, an average of 10 tetheredargatroban molecules were conjugated to each RSA molecule.

Platelets were purified from freshly drawn human blood diluted with theanticoagulant buffer ACD (9 mls of blood to 1 mL of buffer). The bloodwas centrifuged for 25 min at 200×g. The platelet-rich plasma (PRP) wasrecovered with a plastic pipette. The PRP was centrifuged for 15 min at3,000×g in v-shaped plastic tubes. The supernatant, called platelet-poorplasma (PPP), was then removed and saved, and the platelets wereresuspended in buffer Al (35 mM citric acid, 103 mM NaCl, 5 mM each ofglucose and KCl, 2 mM CaCl₂, 1 mM MgCl₃, 3.5 mg/mL BSA, 0.1 μM PGE1 and0.3 U/mL of apyrase at pH 6.5). The resuspended platelets were washed 2times with buffer A1. After the last wash, the platelets wereresuspended in buffer B1 (137 mM NaCl, 2.6 mM KCl, 12 mM NaHCO₃, 0.3 mMNaH₂ PO₄, 3 mM CaCl₂, 1 mM MgCl₂, 5 mM glucose, 3.5 mg/mL BSA and 0.05U/mL apyrase at pH 7.4) at a density of 3×10⁵ /μL. The platelets weremaintained at room temperature until ready for use.

Various parameters relevant to platelet aggregation were obtained on afour channel aggregometer manufactured by AFFI BIO. In brief, thereaction chambers are warmed to 37° C. and are agitated at a rate of1,200 rpm. Light is passed through the reaction chambers, with 300 μL ofbuffer B1 used to set the 100% transmittance level for each chamber.From a series of standardization experiments, it was determined that 0.5U/mL of human thrombin (Sigma) is an optimal aggregating concentrationof thrombin and is used to set the 0% transmittance for the instrument.Standard conditions for the assay involve incubating 250 μL of theplatelet suspension with or without 50 μL of inhibitor diluted in bufferin the reaction chambers for 60 seconds at 37° C. The value oftransmittance is now set as 0%. After this measurement, thrombin isadded to the reaction chambers (20 μl) and transmittance is measured forthe next 6 minutes. Samples were performed in triplicate.

FIG. 11 depicts the delay in thrombin-induced platelet aggregationaffected by various titrations of RSA. By contrast to the sham-treatedRSA, the argatroban-C6-RSA clearly delayed platelet aggregation.

Table 1 tabulates the estimated concentrations of the conjugates whichinhibit 50% of the aggregation (IC₅₀).

                  TABLE 1                                                         ______________________________________                                        Compound           IC.sub.50                                                  ______________________________________                                        Free argatroban       0.1-0.15 μM                                          RSA-argatroban-C6 conjugate                                                                         4.5-7.5  μM                                          RSA sham              >150     μM                                          ______________________________________                                    

As shown in Table 1, using an average of 10 argatrobans/RSA and theknown dilution of RSA in the samples, the estimated IC₅₀ value for theargatroban-C6-RSA was determined to be in the range of 4.5 to 7.5 μM. Bycontrast, the sham-treated RSA did not interfere with plateletaggregation even at 150 μM. Higher concentrations were not tested, sothe IC₅₀ for the sham RSA was not determined. The IC₅₀ for argatroban inthis assay is approximately 0.1 μM, which is in line with literaturevalues. This value is at least an order of magnitude more effective thanthe argatroban-C6-RSA conjugate. This trend was expected based on theincreased IC₅₀ observed for the C6-tethered argatroban carboxylate ascompared to argatroban in the earlier-described K_(m) -based fluorogenicthrombin inhibition assay.

It is evident from the above results that the subject invention providesfor greatly improved treatment involving thrombin inhibition. By use ofthe subject invention, the conjugated thrombin inhibitors maintain forextended periods of time, so that repetitive dosages are not required,compliance by the patient is not required, and protection is ensured.The derivatized thrombin inhibitors of the present invention covalentlyattach to erythrocytes, plasma proteins and various other vascularcomponents while retaining biological activity and are not immunogenic.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A compound of the formula: ##STR2## wherein: Y isa linking group of from 2-30 atoms; andZ is a chemically reactive groupcapable of reaction with a reactive functionality of a target moleculein an aqueous system to form covalent bonds therewith or an activatableprecursor to said chemically reactive group; and wherein said compoundpossesses thrombin inhibitory activity in vivo when bonded to a longlived blood component.
 2. The compound according to claim 1 wherein Y isan alkylene, oxyalkylene or polyoxyalkylene group.
 3. The compoundaccording to claim 1 wherein Y is an alkyl group.
 4. The compoundaccording to claim 3 wherein said alkyl group has 6 carbon atoms.
 5. Thecompound according to claim 3 wherein said alkyl group has 12 carbonatoms.
 6. The compound according to claim 1 wherein Z is a carboxygroup, a carboxy ester group or a mixed anhydride.
 7. The compoundaccording to claim 6 wherein Z is selected from the group consisting ofN-hydroxysuccinimide, isocyanate, thiolester, thionocarboxylic acidester, imino ester, carbodiimide anhydride, carbonate ester andphosphoryl ester.
 8. The compound according to claim 1 wherein Z isN-hydroxysuccinimide.
 9. A composition comprising the compound of claim1 and a physiologically acceptable carrier.
 10. A method for inhibitingthrombin activity in vivo, said method comprising:introducing into thebloodstream of a mammalian host a compound according to claim 1 in anamount sufficient to provide an effective amount for thrombininhibition; wherein said compound reacts with and becomes covalentlybound to long lived blood components and thrombin inhibition ismaintained over an extended period of time as compared to the lifetimeof unbound thrombin inhibitor.
 11. A method according to claim 10wherein said introducing is intravascularly.
 12. A method for inhibitingthrombin activity in vivo, said method comprising:(a) isolating blood orblood components from a mammalian host; (b) contacting ex vivo saidblood or blood components with a compound according to claim 1, whereinsaid compound covalently bonds to one or more reactive functionalitiespresent in said blood or blood components; and (c) introducing into thebloodstream of said mammalian host the covalently bound blood or bloodcomponents obtained in step (b); wherein thrombin inhibition ismaintained over an extended period of time as compared to the lifetimeof unbound thrombin inhibitor.
 13. A method according to claims 10 or12, including the additional steps of:removing a blood sample from saidmammalian host; and analyzing for the presence of said compound bound tolong lived blood components.
 14. A blood portion comprising a compoundaccording to claim 1 covalently bound to at least one long lived bloodcomponent.
 15. A blood portion according to claim 14, wherein said longlived blood component is immunoglobulin.
 16. A blood portion accordingto claim 14, wherein said long lived blood component is erythrocytes.17. A conjugate comprising a compound according to claim 1 covalentlybound to a blood component selected from the group consisting of serumalbumin, immunoglobulin and erythrocytes.
 18. The conjugate according toclaim 17 wherein said blood component is serum albumin.